This invention relates to closures and containers for use in chemistry, biology and biotechnology as well as any other fields which require gas exchange during mixing processes. In particular, the invention relates to closure devices for microbiological and chemical containers such as flasks and test tubes.
In the fields of chemistry, biology and biotechnology, container closures are used to prevent contamination of the chemical reaction or microorganism solution being reacted, cultivated or stored, from airborne particulates or other contaminants. In addition, closures are used to prevent the escape of the chemicals, particles or microorganisms from the container into the atmosphere, where they could be potentially harmful. Furthermore, such closures preferably allow easy access to the container's contents for the purpose of sampling, exchanging or adding media/reagents, etc.
A further requirement for closures in some applications is the need for gas exchange into the container of interest. For aerobic fermentation, for example, oxygen is required for the growth of microorganisms in a nutrient medium. At the same time, waste gasses such as carbon dioxide are often produced and must be eliminated from the container. Therefore, such closures must allow the passage of oxygen molecules into, and carbon dioxide molecules out through, the closure while maintaining appropriate conditions inside the container.
Growth rates and rates of subsequent formation of desired metabolites and products, by aerobic microorganisms are often governed by the available supply of dissolved oxygen in the nutrient medium. Since the solubility of oxygen in water is very low, dissolved oxygen often represents the limiting species for the growth rate of microorganisms. The negative consequences of not maintaining an adequate dissolved oxygen level in the nutrient range from mild to severe (Buchs, J., 2001, Introduction to advantages and problems of shaken cultures, Biochemical Engineering Journal, 7, 91-98.) The first potential consequence is a slowdown of metabolism. While culture experiments may yield some useful results, repeatability is difficult because small differences in flask geometry or operating conditions often have greater effects than the experimental variable under study.
A second potential consequence is a changeover to partial anaerobic metabolism. This results in undesirable by-products that are excreted that change pH and inhibit cell growth. Product formation may, or may not, be affected. A third potential consequence may result if product formation is highly sensitive to oxygen supply. For example, glucoamylase production from Saccharomycopsis fibuligera has been shown to exhibit a narrow oxygenation optimum, even though growth of the organism is not as sensitive to oxygen levels. A fourth potential consequence is a complete change of metabolism mechanisms. Several examples of organisms that completely change their metabolism, secreting new secondary products in response to oxygen limitations have been noted (Katzer, W., Blackburn, M., Charman, K., Martin, S., Penn, J., & Wrigley, S., 2001, Scale-up of filamentous organisms from tubes and shake-flasks into stirred vessels. Biochemical Engineering Journal, 7, 127-134). These changes completely obscure the goals of the original culture experiment. A fifth potential consequence occurs for fermentations that require the organism of interest to grow in the presence of a toxic compound. Sufficient energy production (via oxidative respiration) is required to continuously excrete the toxin from the interior of the cell. In this case, oxygen transfer rate limitations lead to significant cell death of the organism being cultured.
Classically, a flask's or container's closure consists of a gauze or cotton plug inserted into the neck, or opening which acts to allow the diffusion-based exchange of gas molecules between the inside and outside of the container, while also preventing contamination of the container's contents from outside particles or microorganisms. Such cotton plug closures are deficient in many respects, including the tendency to fall apart, difficulty in maintaining homogeneous gas exchange between closures and difficulties in re-sterilization. In addition, cotton plug closures offer substantial resistance to gas transfer, thus causing severe limitations to the level of oxygen, or other desired gasses, diffusing into the container of interest.
The background art is characterized by U.S. Pat. Nos. 920,791; 2,287,746; 2,754,931; 2,849,147; 2,918,192; 3,128,899; 3,326,401; 4,027,427; 4,148,619; 4,665,035; 4,797,367; 4,971,219; 5,037,754; 5,180,073; 5,269,431; 5,395,006; 5,578,491; 5,649,639; 5,783,440; 6,170,684; 6,190,913; 6,536,938 and 7,381,559; the disclosures of which patents are incorporated by reference as if fully set forth herein.